Interaction of the MR and GR in the Nucleus and at DNA

Glucocorticoid actions in the brain are mediated by glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). MR and GR bind endogenously circulating glucocorticoids, share a hormone response element in DNA, and are co-expressed in neurons of the hippocampus and hypothalamus. This arrangement suggests MR and GR functionally cooperate in the regulation of gene expression though hitherto poorly described mechanisms. This possibility was previously exampled by the demonstration of MR-GR interactions in vitro, but demonstration of an interacting complex binding DNA in vivo has not been accomplished. We have utilized a unique cell line (3617ChMR) to study MR-GR interactions in vivo. 3617ChMR incorporates a tandem array of the MMTV long terminal repeat (800-1200 GREs) driving viral Harvey-Ras expression. Accumulation of fluorescent GFP-tagged GR and mCherry-MR at this structure is observable microscopically allowing interactions to be studied at chromatinized DNA. Forster resonance energy transfer (FRET) by lifetime measurement, and fluctuation analysis by cross-correlation number and brightness assay (ccN&B) were used to assess MR-GR interactions in living cells. Paired with standard co-immunoprecipitation, these approaches provide evidence for MR-GR interactions in the nucleoplasm and at DNA and reinforce the expectation that such complexes have a transcriptional role. Computational predictions for MR-GR interactions were obtained using the PRISM algorithm. Unexpectedly, these predictions suggested a more diverse potential for MR-GR complexes beyond the simple heterodimer arrangement. Experimental examination of the stoichiometry of the nucleoplasmic complex (N&B method) did not reveal a heterodimer arrangement. Finally, using the array as an indicator of DNA binding, we show GR is cyclically recruited to DNA by pulses of corticosterone application. Conversely the MR, which does not interfere with GR cyclical behaviour, fails to respond to the ultradian pattern of stimulation remaining loaded at DNA during the washout period; albeit undergoing continuous rapid turnover observable by fluorescence recovery after photobleach (FRAP).